The role of hydrogen bonds in the enzymatic activity of serine proteases is currently the subject of much debate. Some researchers propose that a low-barrier hydrogen bond between His and Asp in the Ser-His-Asp catalytic triad stabilizes the transition state, while other researchers contend that such a strong hydrogen bond is neither necessary nor possible in this protein environment. A direct measurement of the His57 N61 H bond length in ec-lytic protease has been carried out using solid-state NMR dipolar-chemical shift (DIPSHIFT) correlation spectroscopy. Our experiments indicate that the N61 of His57 does not participate in a low-barrier hydrogen bond with Asp102 in the catalytic triad, but participates in a standard hydrogen bond. NH bond distances were measured from 15N -1H DIPSHIFT experiments as a function of pH and inhibitor binding for a sample of cx-lytic protease selectively 15N labeled at the N~1 position of His57 and lyophilized from a solution. In all cases, 99% confidence regions are placed on the measurements using a Monte Carlo method of error determination. The measured NH bond length of 1.105 q.028 A for the transition state mimic is not significantly longer than that for the resting enzyme and indicates that His57 participates in a standard hydrogen bond with Asp 02. If a LBHB were formed, the NH bond length would be expected to be greater than 1.2 A, giving a dipolar coupling of less than 7 kHz. Significantly, the NH bond distance is equal (within experimental error) for the low pH (1.105 q.028 A) and the inhibitor-bound (1.102 q.030 A) samples. As the pH increases and the imidazole ring deprotonates, the NH bond length decreases by .025 A.